Structure-guided and phage-assisted evolution of a therapeutic anti-EGFR antibody to reverse acquired resistance

Acquired resistance to cetuximab in colorectal cancers is partially mediated by the acquisition of mutations located in the cetuximab epitope in the epidermal growth factor receptor (EGFR) ectodomain and hinders the clinical application of cetuximab. We develop a structure-guided and phage-assisted evolution approach for cetuximab evolution to reverse EGFRS492R- or EGFRG465R-driven resistance without altering the binding epitope or undermining antibody efficacy. Two evolved cetuximab variants, Ctx-VY and Ctx-Y104D, exhibit a restored binding ability with EGFRS492R, which harbors the most common resistance substitution, S492R. Ctx-W52D exhibits restored binding with EGFR harboring another common cetuximab resistance substitution, G465R (EGFRG465R). All the evolved cetuximab variants effectively inhibit EGFR activation and downstream signaling and induce the internalization and degradation of EGFRS492R and EGFRG465R as well as EGFRWT. The evolved cetuximab variants (Ctx-VY, Ctx-Y104D and Ctx-W52D) with one or two amino acid substitutions in the complementarity-determining region inherit the optimized physical and chemical properties of cetuximab to a great extent, thus ensuring their druggability. Our data collectively show that structure-guided and phage-assisted evolution is an efficient and general approach for reversing receptor mutation-mediated resistance to therapeutic antibody drugs.

of EGFR, thus inhibiting the proliferation of tumor cells 15,16 . The point mutation T790M, an acquired mutation located in the intracellular domain of EGFR, is the most commonly associated with intrinsic resistance as for acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) 17,18 , whereas point mutations in the EGFR ectodomain (ECD) are inclined to disrupt the specific targeting of therapeutic antibodies 19 . Analysis of clinical cell-free DNA (cfDNA) from mCRC patients with a history of anti-EGFR mAb treatment revealed a cluster of EGFR ECD mutations that mediate acquired resistance by attenuating anti-EGFR antibody binding, for example, EGFR ECD mutations at positions V441, S442, R451, I462, S464, G465, K467, K489, I491 and S492 [20][21][22][23][24][25][26] . All the above residues except R451 are located in the binding site of cetuximab and/or panitumumab 24 . S492R and G465R accounted for approximately 20% of clinical acquired resistance cases, and are among the most common point mutations conferring resistance to anti-EGFR antibodies 20,21 . Strategies for reversing point mutationdriven resistance to anti-EGFR antibodies have focused on the employment of new antibody combinations, e.g., MM-151 and Sym004, to target nonoverlapping epitopes, a time-consuming and labor-intensive approach 26,27 .
In this study, we provide a structure-guided and phage-assisted evolution (SGAPAE) approach for the evolution of the parental antibody cetuximab to reverse resistance mediated by EGFR S492R or EGFR G465R , the two most common antibody-resistance point mutations in cetuximab treatment. We use the SGAPAE approach to determine the energy difference between the bound and unbound states of the interface using the Rosetta platform, developing a semirationally designed library of cetuximab mutants with a restricted epitope. The focused library allows us to efficiently identify three cetuximab variants with minimal mutations (Ctx-VY, Ctx-Y104D and Ctx-W52D) to reverse EGFR S492R or EGFR G465R -driven resistance to cetuximab, while maintaining the optimized physical and chemical properties of cetuximab to a great extent and thus ensuring their druggability. The SGAPAE approach thus constitutes an efficient and viable strategy to overcome single mutation-driven drug resistance while maintaining the druggability of the parental antibodies, and should be a promising approach for the evolution of other biologics.

Many acquired EGFR mutations block cetuximab and/or panitumumab binding and confer drug resistance
Recent studies have shown that a number of EGFR ECD mutations may be acquired in patients with metastatic colorectal cancer (mCRC) after cetuximab and/or panitumumab treatment. These EGFR ECD mutations occurred in EGFR domain III (amino acids 334-504) which is the binding epitope for cetuximab and panitumumab (Fig. 1a), mediating acquired resistance to cetuximab and/or panitumumab through attenuating or abolishing antibody binding [20][21][22][23][24][25][26] .
To better investigate the effect of these mutations on cetuximab and/or panitumumab, we evaluated the binding of cetuximab and panitumumab (1, 10, and 100 nmol/L) with NIH3T3 cells expressing wild-type EGFR or mutant EGFR ( Supplementary Fig. 1) by flow cytometry. Several introduced single mutations nearly abolished anti-EGFR antibody binding to EGFR-e.g., S492R, G465R, S464L or V441D mutation significantly reduced the cetuximab binding affinity, and G465R, G465E, S464L or S442R mutation abolished panitumumab binding to EGFR (Fig. 1b, top). Among the mutations in mCRC patients with a history of cetuximab or panitumumab treatment, S492R and G465R appeared to be the two most common point mutations, present in~20% of patients with clinical resistance 20,21 . A retrospective analysis of plasma samples from a phase III trial (ASPECCT) in a large number of colorectal cancer patients revealed that the EGFR S492R mutation developed in 16% of patients in the cetuximab arm (Fig. 1b, bottom) 20 .
High-resolution EGFR/cetuximab structures show a large, flat binding interface primarily comprising CDRL3, CDRH3, and CDRH2 of cetuximab 28 . Both the S492R and G465R mutations are located in the cetuximab binding site (epitope) on EGFR. We performed homology modeling to predict the effect of the S492R and G465R mutations on cetuximab binding to EGFR (Fig. 1c). The crystal structure of the wildtype EGFR/Fab complex for cetuximab (PDB code: 1YY9) revealed that the CDR of cetuximab formed a central cavity. The S492 and G465 residues of EGFR are accommodated into the above central cavity 29 . According to our structural modeling results, the mutated arginine (R492 and R465) with a large side chain (guanidino group), however, could not fit into the small central cavity in the cetuximab CDR, thus resulting in steric clashes between cetuximab and EGFR S492R or EGFR G465R .
Application of the SGAPAE approach efficiently identified cetuximab variants to reverse the resistance conferred by EGFR S492R or EGFR G465R We used the SGAPAE approach to direct the evolution of cetuximab to reverse EGFR S492R -or EGFR G465R -mediated resistance (Fig. 2). Based on the crystal structure of wild-type EGFR bound to cetuximab Fab (PDB code: 1YY9), the Rosetta platform 30 determined crucial interface residues that are essential for re-establishing the EGFR S492R /cetuximab or EGFR G465R / cetuximab interaction through an exhaustive scan of residues on the interface. In this way, we constructed an epitope-focused cetuximab mutant library to improve the efficiency of subsequent phage display.
To facilitate the construction of the focused phage-display library, we used the RosettaScripts protocol 31,32 and the InterfaceAnalyzer 33 module in Rosetta 30 , software designed for protein-protein interaction (PPI) interface analysis, to narrow down the list of residues critical for restoring cetuximab binding with the EGFR mutants. Among the multiple indicators (e.g., dG_separated, packstat), dG_separated, which represents the energy difference between protein interface separation and binding, was chosen to evaluate the impact of mutations on cetuximab affinity. EGFR S492R or G465R mutation significantly decreased the binding affinity of cetuximab to EGFR, resulting from an increased free energy of binding. The predicted ΔΔG values for 100 models of EGFR S492R /cetuximab and EGFR G465R /cetuximab complexes were consistent with the clinical data (ΔΔG > 0) (Supplementary Table 1), which justified the application of the RosettaScripts protocol and InterfaceAnalyzer method for predicting changes in binding energy accompanying EGFR mutations (Fig. 3a). We further chose the InterfaceAnalyzer method to perform subsequent mutation scanning for the interface residues of cetuximab because it is easy to use and has high calculation efficiency.
A total of 2014 and 1957 predictions were conducted for the EGFR S492R /cetuximab (Cluster 1:684, Cluster 2:665, Cluster 3:665) and EGFR G465R /cetuximab (Cluster 1:684, Cluster 2:627, Cluster 3:646) complexes, respectively (Fig. 3b). According to the changes in binding energy (ΔΔG) in the three clusters (Supplementary Tables 2-7; Supplementary Data 1-6), the intersection of the three clusters, indicating crucial residues for affinity restoration, was obtained. Light chain residues (N32, W94 and T96) and heavy chain residues (V50, D58 and Y014) of cetuximab were identified as potential "replaceable" residues for directed evolution of cetuximab toward binding with EGFR S492R. The heavy chain residues V50 and W52 of cetuximab were identified as mutation site candidates to reverse cetuximab resistance conferred by the acquired EGFR G465R mutation. Therefore, we selected the above potent residues in the receptor binding site of cetuximab for further construction of the phage-display mutagenesis library (Fig. 3c).
Another cetuximab variant targeting EGFR G465R was selected from the G465R library and named Ctx-W52D-scFv (W52D, CDRH3) (Fig. 4b). For further characterization, Ctx-VY-scFv, Ctx-Y104D-scFv and Ctx-W52D-scFv were transformed into human IgG1/κ format (Ctx-VY, Ctx-Y104D and Ctx-W52D) and were then transiently expressed in HEK293F cells. The heavy and light chains of each cetuximab variant appeared at approximately 55 and 30 kDa, respectively, under reducing conditions, and the intact cetuximab variants appeared at approximately150 kDa ( Supplementary Fig. 2a). The SDS-PAGE results, together with the sizeexclusion chromatography (SEC) results and dynamic light scattering (DLS) results, indicated that the introduced mutations did not affect the stability or polymerization state of the parental antibody (Supplementary Fig. 2a-c). On this basis, we further combined the W52D mutation with the V50Q/Y104V or Y104D mutations to generate cetuximab variants Ctx-VWY (V50Q and W52D, CDRH2; Y104V, CDRH3) and Ctx-DD (W52D, CDRH2; Y104D, CDRH3) in order to cover these most common EGFR mutations ( Fig. 4b and Supplementary Fig. 3a).

Cetuximab variants restored binding to EGFR G465R or EGFR S492R
The binding of Ctx-VY, Ctx-Y104D, Ctx-W52D, Ctx-VWY and Ctx-DD to wild-type EGFR or EGFR mutants was analyzed by flow cytometry and ELISA. Panitumumab, wild-type and mutant EGFR-ECD-Fc, and cetuximab variants were successfully produced in HEK293F cells and demonstrated to be intact and correctly assembled according to the SDS-PAGE results under reducing and nonreducing conditions ( Supplementary Fig. 3b, c). As shown in Fig. 4c, cetuximab, panitumumab, Ctx-VY and Ctx-W52D showed a similar binding affinity with wild-type EGFR-expressing stable NIH3T3 (WT-EGFR-NIH3T3) cells and WT-EGFR-ECD-Fc. The introduction of Y104D slightly weakened the binding ability of cetuximab with either wild-type EGFR-expressing stable NIH3T3 cells or WT-EGFR-ECD-Fc in comparison with that of the other evolved mutants Ctx-VY and Ctx-W52D. Furthermore, when two mutations, Y104D and W52D (Ctx-DD), or three mutations, V50Q, W52D and Y104V (Ctx-VWY), were introduced at the same time, the binding affinity of the mutants, especially Ctx-DD, for WT-EGFR-NIH3T3 and WT-EGFR-ECD-Fc was greatly reduced. As expected, cetuximab did not exhibit binding with EGFR S492R -expressing stable NIH3T3 cells (S492R-EGFR-NIH3T3), while Ctx-VY, Ctx-Y104D, Ctx-DD and Ctx-VWY exhibited successfully restoration of a strong interaction with S492R-EGFR-NIH3T3 cells similar to that of panitumumab, an anti-EGFR antibody with a binding site different from that of cetuximab. Intriguingly, Y104D resulted in a higher Emax with EGFR S492R -expressing stable NIH3T3 cells and S492R-EGFR-ECD-Fc than panitumumab, Ctx-VY, Ctx-DD and Ctx-VWY. Among all the cetuximab variants, only Ctx-W52D exhibited restoration of strong binding to EGFR-expressing stable cells and for EGFR-ECD-Fc with the G465R mutation, which abolished the binding of both cetuximab and panitumumab. In addition, we tested the binding affinity of Ctx-W52D in the EGFR G465E model.  However, Ctx-W52D could not reverse G465E-driven resistance to cetuximab ( Supplementary Fig. 4a).

VH-Y104 of cetuximab is a critical residue for the EGFR S492R mutation
The Y104 residue in the cetuximab VH domain (VH-Y104) is alteredin both Ctx-VY and Ctx-Y104D. Hence, we performed site saturation mutagenesis for VH-Y104 and Ctx-Y104X (X = A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y) to thoroughly investigate the impact of this residue on the binding affinity of cetuximab for EGFR S492R (Supplementary Fig. 3d). As shown in Supplementary Fig. 4b, Ctx-Y104D, Ctx-Y104C and Ctx-Y104N are the top three high-affinity binders to EGFR S492R for both stable cells and S492R-EGFR-ECD-Fc, which suggests that the size of the residue side chain is not the major mechanism for restoring EGFR S492R binding. In addition, although Ctx-VY showed favorable binding with EGFR WT and EGFR S492R , the binding of Ctx-Y104V to EGFR S492R was weaker than that of the Ctx-Y104D/C/N mutants ( Supplementary Fig. 4b).
Therefore, we further explored the impact of the V50Q mutation (located in CDRH3) on Ctx-VY binding affinity ( Supplementary Fig. 4c). Cetuximab with either Y104V or V50Q showed a decreased affinity for EGFR S492R , indicating that both mutations are essential for cetuximab to achieve high-affinity binding with EGFR WT and EGFR S492R .
Cetuximab variants restored high-affinity binding to EGFR Mut while exhibiting binding kinetics similar to those of cetuximab in the interaction with wild-type EGFR The binding kinetics of cetuximab and the cetuximab variants with WT-EGFR-ECD-Fc and Mut-EGFR-ECD-Fc were quantitively determined by SPR analysis (Fig. 4d and Supplementary  II  IV   I   III   II  IV   I   III   II  IV   I   III   II  IV   I   III   Cetuximab Fig. 2 | Schematic diagram of the SGAPAE approach for directed evolution of cetuximab. The structure-guided and phage-assisted evolution (SGAPAE) approach was applied to develop cetuximab variants for reversing S492R-or G465R-mediated resistance. First, the crystal structure of the wild-type EGFR/cetuximab Fab complex was obtained from the Protein Data Bank (PDB code: 1YY9). Second, the critical residues in the interface were identified by evaluating the interfacial free energy of the known structure of the EGFR/cetuximab Fab complex and determining the energy difference between the bound and unbound states of the interface with the Rosetta platform. Third, an epitope-restricted cetuximab mutant phage-display library with a size of 10 6 -10 7 phages was generated by randomization of identified critical residues (n = 6). Finally, the cetuximab variants that bound to both EGFR WT and EGFR Mut were selected after panning the cetuximab mutant phage-display library. cetuximab (mean value: kon=1.68×10 5 Ms −1 , koff = 7.29 × 10 −5 s −1 ), which accounted for the incomplete restoration of binding affinity for EGFR WT . The interaction of cetuximab with EGFR S492R or EGFR G465R was not detectable by SPR; in contrast, Ctx-VY, Ctx-Y104D, and Ctx-W52D, exhibited major restoration of binding with EGFR S492R (K D = 2.29 ± 1.20 nM; K D = 2.00 ± 1.42 nM) and EGFR G465R (K D = 4.56 ± 1.13 nM) with respect to the cetuximab/EGFR WT interaction (Fig. 4e). The deleterious impact of the S492R and G465R mutations on cetuximab binding affinity was also confirmed by SPR.
Potential structural basis of the binding mechanism of cetuximab variants to S492R-or G465R-mutated EGFR To explore the potential structural basis underlying the binding of the cetuximab variants to EGFR S492R and EGFR G465R , we simulated the structure of the cetuximab Fab variants bound to domain III of EGFR with the S492R or G465R substitution via the protein structure homologymodelling server SWISS-MODEL (Fig. 5a). Because of the minimal mutations in cetuximab CDR, the overall structures of the EGFR S492R / cetuximab Fab variant complex and EGFR G465R /cetuximab Fab variant complex were extremely similar to that of EGFR WT in complex with cetuximab Fab. The restoration of the binding affinity of the cetuximab variants could be attributed to the significant reduction in steric hindrance via the V50Q/Y104V mutation (Ctx-VY) or Y104D mutation (Ctx-Y104D) and possible strong charge accumulation through the Asp-Arg (D-R) salt bridge (Ctx-Y104D versus EGFR S492R ) (Fig. 5a). Regarding the superiority of Ctx-W52D in recognizing EGFR G465R , the Asp-Arg (D-R) salt bridge-mediated reconnection and reduction in steric hindrance probably contributed simultaneously to restoration of the binding ability, since the large indolyl side chain of tryptophan (W) was downsized to the carboxyl group of aspartate (D). We also compared the paratopes of cetuximab and the cetuximab variants in surface mode (Fig. 5b). The size of the central cavity surrounded by CDRL3, CDRH2 and CDRH3 is different between cetuximab and the cetuximab variants. The larger central cavity of the cetuximab variants could accommodate the side chain of mutated R492 or R465, which may explain the molecular mechanism underlying the restoration of cetuximab binding to EGFR S492R and EGFR G465R (Fig. 5b).

The evolved cetuximab variants successfully inhibited EGFinduced EGFR S492R and EGFR G465R activation
The primary mechanism of action of cetuximab is the blockade of ligand-induced EGFR activation and downstream signaling, including    signaling through the PI3K/Akt and MAPK/Erk pathways which have been linked to many processes crucial to tumor progression, including metastasis and cell survival, proliferation, adhesion, differentiation, migration, transformation, and motility 36 . To evaluate the inhibitory activities of the cetuximab variants, we established stable NIH3T3 cells expressing full-length EGFR WT or EGFR Mut as an in vitro cell model.
Robust phosphorylation of EGFR and activation of Akt and Erk in the downstream pathway were induced in stable WT-EGFR-NIH3T3, S492R-EGFR-NIH3T3 and G465R-EGFR-NIH3T3 cells upon EGF stimulation, according to the western blot results (Fig. 5c). Pretreatment with cetuximab, panitumumab, Ctx-VY, Ctx-Y104D, or Ctx-W52D reduced the level of phosphorylated EGFR (pEGFR) to basal levels and decreased the levels of phosphorylated Akt and Erk (pAkt and pErk) in WT-EGFR-NIH3T3 cells (Fig. 5c, left). In the S492R-EGFR-NIH3T3 cells, panitumumab, Ctx-VY and Ctx-Y104D but not cetuximab abolished EGF-induced EGFR phosphorylation and downstream signaling (Fig. 5c, middle), which was consistent with the binding affinity results (Fig. 4c). In G465R-EGFR-NIH3T3 cells, Ctx-W52D significantly reduced EGF-stimulated EGFR G465R activation, although cetuximab and panitumumab did not, according to the western blot results for downstream pathway indicators, such as pEGFR, pAkt and pErk (Fig. 5c, right). Owing to the incomplete restoration of the binding affinity of the cetuximab variants for EGFR WT , the Erk attenuation induced by the cetuximab variants was still limited, particularly in WT-EGFR-NIH3T3 cells.

Cetuximab variants with unimpaired effector activities potently inhibited tumor cell proliferation
Cell proliferation inhibition and immune effector activity are important mechanisms of action for antitumor therapeutic antibodies (Fig. 6a). We further evaluated the in vitro efficacy of cetuximab and the cetuximab variants against the wild-type EGFR cell line SW48 (WT-EGFR) 38 , SW48 cells stably expressing full-length EGFR G465R , i.e., G465R-EGFR-SW48 (WT/G465R-EGFR-Heterozygote), and COLO320DM cell lines stably expressing full-length wild-type or an EGFR mutant (WT-EGFR-COLO320DM, S492R-EGFR-COLO320DM and G465R-EGFR-COLO320DM) (Fig. 6b, c and Supplementary Fig. 6). COLO320DM is an EGFR-negative human colorectal cancer cell line with wild-type KRAS/ BRAF, which is an ideal cell model for generating stable EGFR mutant cell lines. Both cetuximab and the cetuximab variants inhibited the proliferation of SW48 cells and WT-EGFR-COLO320DM cells and exhibited similar in vitro efficacy and wild-type EGFR binding ability (Fig. 6b, left and Fig. 6c, top). Considering the heterogeneous cell surface expression of EGFR and its variants in tumors, we generated the G465R-EGFR-SW48 cell line to evaluate the inhibitory effect of cetuximab and Ctx-W52D. Cetuximab showed lower efficacy in inhibiting EGFR signaling in the G465-EGFR-SW48 model, while Ctx-W52D maintained its high potency. This result indicated that exogenous introduction of full-length EGFR G465R in the SW48 cell model impaired the inhibitory effect of cetuximab but not Ctx-W52D on EGFR downstream signaling (Fig. 6b, right). In the S492R-EGFR-COLO320DM cell model, Ctx-VY and Ctx-Y104D exhibited similar inhibition efficacies; however, cetuximab had almost no inhibitory effect (Fig. 6c, middle). Consistent with its EGFR G465R binding ability, only Ctx-W52D inhibited cell proliferation in a dose-dependent manner in the G465R-EGFR-COLO320DM cell model in comparison with cetuximab and control IgG (Fig. 6c, bottom).
Immune effector activity also plays an important role in the antitumor activity of therapeutic antibodies. We also investigated the complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) activity of cetuximab variants according to previously described methods 39,40 . The results showed that the cetuximab variants exhibited ADCC (Fig. 6d) and CDC (Fig. 6e) activities similar to those of the parental antibody cetuximab in EGFR WT model cells, and showed additional ADCC and CDC activities in EGFR Mut -expressing model cells, benefiting from their restored EGFR Mut binding ability.

Cetuximab variants can inhibit the growth of mutant EGFRpositive and WT-EGFR-positive tumors in vivo
We used colorectal cancer (CRC) cell line-derived xenograft (CDX) models to evaluate the in vivo antitumor activity of Ctx-VY, Ctx-Y104D and Ctx-W52D. In the SW48 (WT-EGFR) xenograft model (Fig. 7a), tumor growth was significantly suppressed in the Ctx-VY, Ctx-Y104D, Ctx-W52D and cetuximab groups compared to the PBS group, and the in vivo antitumor activities of the cetuximab variants were consistent with their binding affinities for EGFR. In particular, the in vivo performance of Ctx-W52D and cetuximab was highly similar according to the mean tumor volume and tumor weight data (Fig. 7b, c). No body weight loss was observed in mice during cetuximab and cetuximab variants treatment (Fig. 7d). In addition, we could not detect any obvious tissue damage in the important organs, such as heart, liver, spleen, lung, and kidney, in mice, according to histological examination of tissue sections via H&E staining (Supplementary Fig. 7a). Further serological studies revealed that serum ALT, AST, BUN and Cr did not show any significant difference among the vehicle and treatment groups ( Supplementary Fig. 7b), excluding the possibility of severe liver or kidney toxicity. In the G465R-EGFR-SW48 (WT/G465R-EGFR-Heterozygote) xenograft model (Fig. 7e), cetuximab could not bind with EGFR G465R or affect EGFR G465R signaling, therefore the exogenous introduction of EGFR G465R in SW48 lowered the potency of cetuximab on tumor growth inhibition in the G465R-EGFR-SW48 (WT/ G465R-EGFR-Heterozygote) model, in comparison to the SW48 (WT-EGFR) model. On the other hand, the Ctx-W52D, capable of binding with EGFR WT and EGFR G465R , showed similar in vivo efficacy in two xenograft models (Fig. 7f, g). No body weight loss was observed in mice during cetuximab and Ctx-W52D treatment (Fig. 7h). In addition, cetuximab and its variants showed similar in vivo antitumor activity in the WT-EGFR-COLO320DM xenograft model (Fig. 7i), with respect to the SW48 (WT-EGFR) model (Fig. 7j-l). The in vivo efficacy of Ctx-VY and Ctx-Y104D was maintained in the S492R-EGFR-COLO320DM Fig. 5 | The mechanisms of action by which the cetuximab variants in inhibit EGFR S492R or EGFR G465R activation. a The modeled structural mechanisms by which the cetuximab variants restore binding with EGFR S492R or EGFR G465R . The critical residues in the cetuximab variants and the EGFR mutations are highlighted as colored sticks in the interface. The structures of VH domain in the cetuximab/ cetuximab variants and EGFR S492R or EGFR G465R ECD domain III are shown in white and gray, respectively. b The central cavities (circled in black) in the paratopes of cetuximab (PDB code: 1YY9) and its variants. c The mechanism by which the cetuximab variants inhibit the ligand-induced activation of EGFR S492R or EGFR G465R , as shown by western blot analysis. Stable NIH3T3 cells were stimulated with EGF (0.5 nM) and immunoblotted for total and phosphorylated EGFR, Akt, and Erk (pEGFR Y1068 , pAkt S473 , and pErk T202/Y204 ). d The mechanism by which the cetuximab variants inhibit the ligand-induced receptor internalization and degradation of EGFR S492R or EGFR G465R , as shown by flow cytometry. The mean fluorescence intensity (MFI) of different stable HEK293T cells was determined after 48 h of preincubation with the indicated concentrations of antibodies. MFI values were normalized to the EGFR-eGFP signal in cells with no antibody treatment. All above assays were performed in triplicates. The bars indicate the mean ± SD values. Source data are provided as a Source Data file. xenograft model; however, there was no significant difference between the cetuximab group and the PBS group in tumor growth suppression (Fig. 7m-o). No body weight loss was observed in mice during Ctx-VY and Ctx-Y104D treatment (Fig. 7p). The in vivo efficacy of Ctx-W52D was also maintained in the G465R-EGFR-COLO320DM xenograft model; however, there was no significant difference between the cetuximab group and PBS group in tumor growth suppression (Fig. 7q-s). No body weight loss was observed in mice during Ctx-W52D treatment (Fig. 7t). Further H&E staining results demonstrated that the tumor tissue morphology was severely disrupted in the cetuximab variant groups compared to the cetuximab group and PBS group (Supplementary Fig. 8a). Immunostaining for EGFR showed that intratumoral EGFR WT expression was downregulated after the treatment of cetuximab and its variants in the EGFR WT model. However, in the EGFR Mut models, immunostaining for EGFR showed that intratumoral EGFR Mut expression was downregulated in the cetuximab variant groups, while no difference was observed between the cetuximab group and vehicle group (Supplementary Fig. 8a). Immunohistochemical staining for Ki-67, an indicator of tumor progression, revealed markedly fewer intratumoral Ki-67-positive cells in cetuximab variant groups, indicating that the cetuximab variants effectively inhibited EGFR WT and EGFR Mut tumor cell proliferation in the xenograft model ( Supplementary Fig. 8a-d). The TUNEL assay revealed that there were apoptotic tumor cells in cetuximab variant groups, further confirming the in vivo efficacy of the cetuximab variants against EGFR WT and EGFR Mut tumor cells ( Supplementary Fig. 8a, e-g).

Discussion
Owing to deleterious single point mutations in the ectodomain of EGFR, such as S492R and G465R, acquired resistance to anti-EGFR therapeutic antibodies emerges during clinical antibody therapy, hindering further applications of antibody derivatives, including antibody-drug conjugates (ADCs) 41 and bispecific antibodies (bsAbs) 42 . In this study, we successfully applied a structure-guided and phageassisted evolution (SGAPAE) approach to direct the evolution of cetuximab, successfully reversing the acquired drug resistance conferred by EGFR ECD mutations. According to structural analysis and computational prediction of EGFR ECD with the S492R mutation, we identified 6 critical residues in the CDRs of cetuximab and created a 6-residue randomized phage-display library. After panning the epitope-restricted cetuximab mutant library, the cetuximab variants Ctx-VY and Ctx-Y104D were identified to exhibit restored binding affinity for EGFR S492R while maintaining their binding affinity for EGFR WT . The SGAPAE approach also reversed EGFR G465R -driven drug resistance to cetuximab, demonstrating its more general application. Another cetuximab variant, Ctx-W52D, with a single mutation in the cetuximab CDR, was shown to exhibit restored binding affinity for EGFR G465R . Intriguingly, all cetuximab variants maintained the functions of their parental antibody cetuximab, including blockade of EGFR activation and downstream signaling, induction of EGFR degradation, inhibition of cell proliferation, and CDC and ADCC activity. Collectively, these results demonstrate that with the SGAPAE approach, we efficiently achieved epitope-restricted evolution of cetuximab and identified cetuximab variants with minimal point mutations (1 or 2) to reverse EGFR S492R -or EGFR G465R -mediated drug resistance.
The application of SGAPAE in cetuximab evolution, which involves the use of the Rosetta platform, relies heavily on the structural information of the EGFR/cetuximab complex. Developing a computational model consistent with the binding characteristics of an antibody/antigen complex is essential for the prediction of potential hot spot residues for evolution. The Rosetta platform offers several advantages for evaluating the interfacial free energy. The Inter-faceAnalyzer module of Rosetta determines the energy difference between the bound and unbound states of the interface, which is beneficial for evaluating the effect of a single point mutation on the affinity of cetuximab based on the known structure of the EGFR/ cetuximab Fab complex. The flexible setting of constraints (e.g., residue distance) in the Rosetta platform facilitates fast and exhaustive searching surrounding the point mutation. Finally, with the restricted binding site of the cetuximab variant, Rosetta provides the most accurate local docking pose after simply orienting the Fab in the expected binding site, leading to the identification of key amino acid residues for randomization. Therefore, the Rosetta platform in the SGAPAE approach was ideally suited to address EGFR single mutation (e.g., S492R or G465R)-driven acquired resistance to cetuximab and should be effective for addressing single mutations in other receptors (e.g., HER2 S310F, which abolishes pertuzumab binding) 43 .
To date, phage and yeast display technologies have been widely used in antibody discovery 44 and cytokine engineering, e.g., IL-2 bias screening 45 . However, to the best of our knowledge, phage display has not been applied to overcome single mutation-mediated antibodydrug resistance. Because of the limited key residues for randomization, the size of the cetuximab mutant library was reduced significantly to 10 6 -10 7 phages, which exhibited superiority over conventional phagedisplay libraries with a size of 10 9 -10 12 phages in biased screening processes 46 . The diversity of the semirationally designed cetuximab mutant library is confined within the binding epitope of the parental cetuximab structure, providing an obvious evolutionary direction toward EGFR and thereby retaining the highly similar binding site in the selected cetuximab variants. In combination with the known structural information and computational predictions, our SGAPAE approach was able to improve the screening efficiency for effective cetuximab variants. The binding epitope of the parental antibody is usually correlated with antibody efficacy. With the epitope-restricted cetuximab mutant phage-display library, we found that minimal point mutations or even a single point mutation (e.g., Y104D or W52D) in the cetuximab CDR could completely reverse EGFR S492R -or EGFR G465Rmediated cetuximab resistance without altering the binding epitope or attenuating antibody efficacy. In addition, the evolved cetuximab variants (Ctx-VY, Ctx-Y104D and Ctx-W52D) with one or two amino acid substitutions in the CDR, are less likely than other variants to perturb the rest of the antibody domains, inheriting the optimized physical and chemical properties of cetuximab to the greatest possible extent.
In summary, we successfully applied the SGAPAE approach to efficiently reverse EGFR S492R -or EGFR G465R -mediated resistance to cetuximab. Acquired drug resistance during antibody therapy has greatly undermined the effectiveness of antibody treatment due to frequent mutations in the target receptors (e.g., EGFR and HER2). The SGAPAE approach provides an efficient and viable strategy to overcome single mutation-driven drug resistance while maintaining the druggability of parental antibodies. The SGAPAE approach also constitutes a promising strategy for efficiently evolution of other proteins, such as recombinant enzymes and cytokines, whose structural information is usually abundant and rapidly expanding owing to the rapid development of structural biology.

Ethical statement
Experiments were carried out according to the National Institutes of Health (United States) Guide for the Care and Use of Laboratory Animals. All animal work was performed in accordance with the protocol Fig. 6 | The cetuximab variants inhibited the growth of wild-type EGFR-and S492R/G465R-mutated EGFR-positive cells in vitro. a The mechanisms of action underlying the antitumor activities of cetuximab. b The in vitro efficacy of cetuximab and the cetuximab variants in inhibiting the proliferation of cetuximab-sensitive/resistant cells. The wild-type EGFR cell line SW48 (WT-EGFR) and SW48 cells stably expressing full-length EGFR G465R (G465R-EGFR-SW48) were used as model cell lines, and treated with serial concentrations of antibodies for 3 days. Cell viability was determined by a CCK-8 assay. c The in vitro efficacy of cetuximab and the cetuximab variants in inhibiting the proliferation of cells in the established CRC xenograft model. WT-EGFR-COLO320DM cells, S492R-EGFR-COLO320DM cells and G465R-EGFR-COLO320DM cells were used as model cell lines, and treated with serial concentrations of antibodies for 3 days. Cell viability was determined by a CCK-8 assay. d The ADCC activity of cetuximab and the cetuximab variants. LDH release was detected after hours of coincubation of PBMC effector cells and WT-EGFR-eGFP-HEK293T or mutant-EGFR-eGFP-HEK293T target cells at an E:T ratio of 40:1. e The CDC activity of cetuximab and the cetuximab variants. WT-EGFR-NIH3T3 cells and mutant-EGFR-NIH3T3 cells were incubated with serial concentrations of antibodies in the presence of 10% human serum for 2 h, and cell viability was measured by a CCK-8 assay. All above experiments were performed in triplicates. The bars indicate the mean ± SD values (*p < 0.05, **p < 0.01, N.S. means not significant; two-tailed Student's t-test   Mice BALB/c nude mice (6-8 weeks old, male, body weight 20-30 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and housed under specific pathogen-free conditions. Experiments were carried out according to the National Institutes of Health (United States) Guide for the Care and Use of Laboratory Animals. All animal work was performed in accordance with the protocol approved by the Committee on the Ethics of Animal Experiments of Zhejiang University (Hangzhou, China).

Homology modeling and structural analysis
The online server SWISS-MODEL was used to predict the 3D protein structures of S492R-and G465R-mutated EGFR and the cetuximab variants using the crystal structure of the wild-type EGFR/Fab complex for cetuximab (PDB code: 1YY9) as a template. Essential amino acid substitutions that lead to loss of cetuximab or panitumumab binding to EGFR were identified according to the literature (Refs). All predicted structures were decorated and displayed with the open-source version of the PyMOL program (https://github.com/schrodinger/PyMOL -open-source).

Structure preparation and computational mutation scanning
The crystal structure of the wild-type EGFR ECD/cetuximab Fab complex was obtained from the Protein Data Bank (PDB code: 1YY9). Waters and other HET residues (e.g., NDG, NAG, BMA) in the crystal structure were removed. Missing loops and hydrogen atoms were added to the cleaned structure of the antibody/antigen complex, which was then subjected to initial refinement using the Relax application in Rosetta. The relaxed structure with the lowest energy was selected for subsequent mutant modeling. The change in binding energy (ΔΔG) caused by EGFR point mutation was used as a criterion for method suitability testing. One hundred structural models were generated for each of the EGFR S492R / cetuximab and EGFR G465R /cetuximab complexes, and their corresponding ΔΔG values (see Eq. 1) were simultaneously calculated via a RosettaScripts protocol using the ref2015 energy function. In addition, the InterfaceAnalyzer application was used to determine the difference in binding energy (dG_separated) between the EGFR mutants and wild-type EGFR.
All predicted structures of EGFR S492R /cetuximab and EGFR G465R /cetuximab were clustered according to the interface residues between the antigen and antibody. Three representative structures were selected for each EGFR S492R /cetuximab and EGFR G465R /cetuximab complex. For cetuximab's amino acids within 5 Å of any residue of EGFR, a systematic single point mutation scan was performed by using Rosetta's pmut_scan application. The binding energies of all EGFR/cetuximab mutant and EGFR/cetuximab wild-type complexes were calculated by using the InterfaceAnalyzer application. Finally, cetuximab variants potentially effective against EGFR mutants were identified based on the change in binding energy (see Eq. 2).
Generation of stable cell lines To generate cell lines with stable expression of EGFR and its mutants, the sequence encoding N-terminal Flag-tagged full-length human wildtype EGFR was inserted into the expression vector pCMV3-hygromycin/ampicillin (Sino Biological, China). S492R-, I491M-, K489E-, K467T-, G465R-, G465E-, S464L-, I462R-, R451C-, S442R-and V441Dmutated EGFR constructs were generated by site-directed mutagenesis using the wild-type EGFR construct as a template. Primers used in this experiment were listed in Supplementary Table 9. NIH3T3 cells were transfected with the wild-type or EGFR mutant constructs by a Lipofectamine 3000 transfection kit according to the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA) and were then treated with hygromycin B (300 μg/mL; Solarbio, China) for 96 h. After immunostaining with a mouse polyclonal anti-Flag tag antibody (Genscript, China) at a dilution of 1:500 and a FITC-conjugated goat anti-mouse IgG (H + L) secondary antibody at a dilution of 1:500, stable cells expressing wild-type or mutant EGFR were sorted by fluorescenceactivated cell sorting (FACS) in a MoFlo Astrios EQ flow cytometer (Beckman, USA). Single NIH3T3 cell clones that stably expressed wildtype or mutant EGFR (S492R, I491M, K489E, K467T, G465R, G465E, S464L, I462R, R451C, S442R, or V441D) were identified with an ACEA NovoCyteTM flow cytometer (ACEA Biosciences, USA). Stable HEK293T cells harboring C-terminal eGFP-tagged human wild-type, S492R-mutated or G465R-mutated EGFR were generated as described above. Stable SW48 cells overexpressing G465R-mutated EGFR and stable COLO320DM cells overexpressing wild-type, S492R-mutated or G465R-mutated EGFR were also generated as described above.

Recombinant EGFR-ECD-Fc fusion protein
The fusion gene fragment of the human wild-type EGFR ectodomain (ECD, residues 25-644) and the human IgG1 Fc-fragment were cloned into the expression vector pMH3. The S492R-EGFR-ECD-Fc, G465E- EGFR-ECD-Fc and G465R-EGFR-ECD-Fc mutant constructs were generated by site-directed mutagenesis. Primers used in this experiment were listed in Supplementary Table 9. The recombinant EGFR-ECD-Fc fusion protein and mutants were expressed in HEK293F cells through transient transfection with polyethylenimine (PEI). Recombinant fusion proteins were purified from culture supernatants using a HiTrap Protein A column (GE Healthcare, PA, USA) in an ÄKTA pure system (GE Healthcare, PA, USA), dialyzed against PBS (pH 7.4) and stored in −80°C for further study.

Generation of phage-display libraries
The single-chain variable fragment (scFv) of cetuximab was cloned into the SfiI-NotI restriction sites of the phagemid vector pCANTAB-5E. On the basis of computational prediction, the S492R-associated library was constructed with random mutations in CDR2 and CDR3 of the VH domain (targeting Val50, Asp58; Tyr104), and CDR1 and CDR3 of the VL domain (targeting Asn32; Trp94, Thr96). The G465R-associated library was generated with random mutations in CDR2 and CDR3 of the VH domain (targeting Val50, Trp52). These residues were mutated to 20 other amino acids by introducing NNS (N = A, T, C or G; S = C or G) codons in the oligonucleotide primer, and the corresponding scFv gene fragments were amplified by PCR. Primers used in this experiment were listed in Supplementary . The percentages of monoclonal antibody-stained cells among total EGFR-positive cells were determined. The mean fluorescence intensity (MFI) of the immunostained cells was also determined by flow cytometry. FACS sequential gating strategy is shown in Supplementary Fig. 9a. To evaluate the binding ability of the cetuximab variants, wild-type EGFR, EGFR S492R , EGFR G465E or EGFR G465R -expressing NIH3T3 cells were treated with serial concentrations of cetuximab, panitumumab or the cetuximab variants, and were then fluorescently labeled with the FITC-conjugated goat anti-human IgG (H + L) secondary antibody (Beyotime, China) at a dilution of 1:500 before flow cytometric analysis. FACS sequential gating strategy is shown in Supplementary Fig. 9b. Data were analyzed by GraphPad Prism software.

Antibody binding ability with EGFR-ECD
The binding of cetuximab, panitumumab, and the cetuximab variants to EGFR-ECD was analyzed by ELISA. Ninety-six-well microtiter plates were coated with WT-EGFR-ECD-Fc, S492R-EGFR-ECD-Fc, G465E-EGFR-ECD-Fc, and G465R-EGFR-ECD-Fc at a concentration of 1 µg/mL overnight at 4°C. Antibodies were diluted in PBS with 5% (w/v) nonfat milk and added to the coated plates at concentrations ranging from 1 × 10 −4 nM to 100 nM. The plate wells were washed five times with PBST. Antibodies were detected with an HRP-conjugated polyclonal goat anti-human kappa light chain antibody (Thermo Fisher Scientific, USA) at a dilution of 1:500 using TMB as the substrate.

Surface plasmon resonance
Antibody binding affinities were determined by a NeoSPR-M100 instrument (Neoline, Hangzhou, China). Flow cells composed of carboxyl chips (Neoline, Hangzhou, China) were coupled with recombinant WT-EGFR-ECD-Fc, S492R-EGFR-ECD-Fc and G465R-EGFR-ECD-Fc through amine-coupling chemistry, and the coupling buffer PBS was used as a reference. Cetuximab or the cetuximab variants were injected over the immobilized WT-EGFR-ECD-Fc, S492R-EGFR-ECD-Fc or G465R-EGFR-ECD-Fc (coupled at densities ranging from 96-293 relative units to flow cells composed of carboxyl chips) at a flow rate of 20 µL/min at 25°C in PBS. Flow cells were regenerated following each injection using 30 mM NaOH buffer. Kinetic parameters were analyzed by TraceDrawer software.

Induction of EGFR degradation
Antibody-induced EGFR-eGFP degradation was assessed by flow cytometry. HEK293T cells overexpressing WT-EGFR-eGFP, S492R-EGFR-eGFP and G465R-EGFR-eGFP were seeded in 48-well plates overnight in a 37°C incubator with 5% CO 2 , and were then treated with cetuximab, panitumumab, Ctx-VY, Ctx-Y104D, Ctx-W52D or control IgG for 48 hours. Mean fluorescence intensity (MFI) values were normalized to those in the untreated controls. FACS sequential gating strategy is shown in Supplementary Fig. 9c. Data were analyzed by GraphPad Prism software.

Inhibition of cell proliferation
SW48, G465R-EGFR-SW48 and COLO320DM cells stably overexpressing wild-type or mutant EGFR were seeded in 96-well plates overnight in a 37°C incubator with 5% CO 2 and treated with serial concentrations of therapeutic antibodies or cetuximab variants. Three days post treatment, cell viability was evaluated using a Cell Counting Kit-8 (CCK-8, Japan) and measuring the absorbance at 450 nm in a Model 680 microplate reader (Bio-Rad, USA). The cell proliferation inhibition rate is shown relative to cells treated with PBS only. Data were analyzed in GraphPad Prism software.

ADCC and CDC assays
For the antibody-dependent cellular cytotoxicity (ADCC) assay, ten thousand target cells were coincubated with 5×10 5 human peripheral blood mononuclear cells (PBMCs) for 10 hours, and cell viability was determined by a lactate dehydrogenase (LDH) detection kit (LDH Cytotoxicity Assay Kit; Beyotime). The percentage of specific lysis was determined as follows: % lysis = (experimental LDH releasespontaneous LDH release)/(maximum LDH release − spontaneous LDH release) × 100. All assays were performed in triplicates. The results are presented as the mean ± SD values. For the complement-dependent cytotoxicity (CDC) assays, antibodies were incubated with 1 × 10 4 target cells/well and 10% human serum for 2 hours. Cell viability was determined by a Cell Counting Kit-8 (CCK-8, Japan). The percentage of specific lysis was determined as follows: % lysis = (experimental lysisnonspecific lysis) × 100. Experiments were performed in triplicate. The results are presented as the mean ± SD values.

In vivo efficacy of antibodies in mouse xenograft models
Mouse xenograft models bearing EGFR WT -or EGFR Mut -positive cells were generated by subcutaneous inoculation of 5 × 10 6 SW48 (WT-EGFR), G465R-EGFR-SW48 (WT/G465R-EGFR-Heterozygote), WT-EGFR-COLO320DM, S492R-EGFR-COLO320DM or G465R-EGFR-COLO320DM cells into the right flanks of nude mice. When the mean tumor volume reached~50 mm 3 , the mice were randomly divided into 20 groups (PBS, cetuximab, Ctx-VY, Ctx-Y104D and Ctx-W52D; n = 5 mice per group). Cetuximab or the cetuximab variants were injected intravenously into xenografted mice via the tail vein at a dosage of 25 mg/kg, once every 3 days for four times (q3d×4). Mouse body weights were measured once every 2-3 days with an electronic balance, and the tumor size was measured every 2-3 days with an electronic caliper; the tumor volume was calculated using the formula (L × W × W)/2, in which L represents the larger diameter of the tumor and W represents the smaller diameter. Tumor tissues were collected for preparation of paraffin sections for the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining assay, immunohistochemical staining (EGFR and Ki-67), and hematoxylin and eosin (H&E) staining. Organs, including the heart, liver, spleen, lung, and kidney, were isolated for preparation of paraffin sections for H&E staining. Serum was collected for measurement of ALT, AST, BUN, and Cr to evaluate liver and kidney function. A Cobas c311 analyzer (Roche Diagnostics GmbH, Germany) was used to test ALT, AST, BUN and Cr.

Immunohistochemical staining
Tumor tissues were fixed with 10% formaldehyde and embedded in paraffin. After dewaxing, rehydration, and antigen retrieval, sections were blocked with goat serum blocking solution and were then stained using rabbit polyclonal anti-EGFR (1:200, Beyotime, AF5153) or rabbit polyclonal anti-Ki-67 (1:500, Abcam, ab15580) antibodies overnight at 4°C. After staining with the HRP-conjugated goat anti-rabbit IgG (H + L) antibody (Zhongshan Goldbridge Biotechnology, PV6001, 100 μL per sample) at 37°C for 30 min, tumor tissue sections were visualized by a DAB detection kit (Boster Biological Technology, AR1026) and an Olympus BX61 light microscope (Olympus, Shinjukuku, Tokyo, Japan). For detection of apoptosis, paraffin-embedded sections of tumor tissues were labeled by a POD-TUNEL Kit (Roche Inc.) according to the manufacturer's instruction. All images were acquired by an Olympus BX61 light microscope. Quantification was performed at ×400 magnification (3 images per tumor) using ImageJ 1.52a software.

Statistics and reproducibility
The statistical analysis is described in the "Methods" section or figure legends. All the experiments were performed at least in triplicate and repeated.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
The structures of wild-type EGFR ECD/cetuximab Fab complex was obtained from the Protein Data Bank (PDB code: 1YY9). All data generated or analyzed during this study are included in this article, its Supplementary